I. Antibiotic Use and Antibiotic-Resistance
The term "antibiotic" is broadly defined as a chemical compound produced by one microorganism that inhibits the growth of a different microorganism. Today, there are more than 150 antibiotics which are classified by their chemical structures and mechanisms of action, examples of which are listed in Table 1 below (Lawson et al, The American Biology Teacher, 6:412-417 (1998)).
TABLE 1 ______________________________________ Chemical Class and Mechanism of Action of Common Antibiotics Antibiotic Class Mechanism of Action ______________________________________ Ampicillin Broad-spectrum Inhibits cell wall penicillin synthesis Ceftriaxone 3.sup.rd generation Inhibits cell wall cephalosporin synthesis Erythromycin Macrolide Inhibit protein synthesis Norfloxacin Quinolone Inhibits DNA synthesis Streptomycin Aminoglycoside Inhibits protein synthesis Sulfisoxazole Sulfonamide Competitive enzyme inhibitor Tetracycline Tetracycline Inhibits protein synthesis ______________________________________
When the first therapeutic antibiotic, penicillin, was introduced in the early 1940's, many believed that the threat from infectious diseases was over. However, in the past 25 years, through the abuse and misuse of antibiotics, many bacteria have developed resistance to these antibiotics. Today, there are strains of virtually every major bacterial human pathogen that are resistant to some of the most effective antibiotics. These strains include pathogens that can cause diarrhea, urinary tract infections, otitis media, meningitis, tuberculosis, gonorrhea, pneumonia, dysentery, wound infections, septicemia, bacteremia and surgical infections (Lippe, Breakout: The Evolving Threat of Drug-Resistant Diseases, Sierra Clubs, San Francisco (1995)).
Thus, while 90% of bacterial infections are successfully treated with first line antibiotics, there are increasingly situations in which over 40% of the infections are resistant to one or more antibiotics (including second line products). The newest antibiotic, vancomycin, has been shown to be the only antibiotic that is effective against some pathogenic bacteria. It has become the last line of defense against some infections, particularly those by methicillin-resistant Staphylococcus aureus.
In addition, some less pathogenic strains of the genus Enterococcus have vancomycin-resistance genes, and have been shown, in the laboratory, to transfer this resistance to Staphylococcus strains (Lawson et al, supra). As a result, the physician will have no treatment for infections by these Staphylococcus strains.
The mechanisms of bacterial resistance to antibiotics include the following:
(1) Loss of cell permeability to the antibiotic; PA1 (2) Enzymes that render the antibiotic ineffective; PA1 (3) Export of the antibiotic out of the cell once it enters the cell; PA1 (4) Modification of the target of the antibiotic; and PA1 (5) Modification of metabolic pathways which result in by-passing the reaction inhibited by the antibiotic. PA1 3494 B.C.: The initial discovery of herbal medicine by emperor Shen Nong; PA1 500 B.C.: The flowering of Chinese medicine: medicine, religion, ethics, philosophy; and PA1 16th Century: Li Shizhen (1517-1593) writes Outlines and Divisions of Herbal Medicines.
The seriousness of these mechanisms of antibiotic resistance is accentuated by the ability of the bacteria to transfer the resistance to other microorganisms, some of which may be fairly genetically unrelated to the antibiotic-resistant strain. The antibiotic-resistance transfer can occur through one of the following mechanisms: conjugation, transduction, transformation or transposition.
Antibiotic-resistant microorganisms add an estimated $200 million/year to medical bills. When costs for extended hospital stays are considered, the estimated costs increase by $30 billion/year (Phelps, Medical Care, 27:194-203 (1989)). Thus, there is a crucial need for novel antimicrobial agents which can effectively inhibit the growth of bacteria by mechanisms different from those of existing antibiotics.
II. Traditional Chinese Medical Herbal Formulations
The historic milestones in Traditional Chinese Medicine (TCM) are as follows:
The ancient Chinese understood the value of a combinatorial approach to the treatment of diseases. They believed that a disease can, and often, affects more than a single function and thus, treatment must be directed to multiple targets. A mixture of different herbs was thus, designed to neutralize the multiple effects of a disease. The Western "silver bullet" approach of one single chemical to cure one disease in all patients has not been readily accepted in TCM.
Most formulations of TCM contain 6 to 12 herbs. Throughout the history of TCM, there have been many different methods of classifying the ways in which medicinal substances can be combined (Liao Zhong-Chun, Annotated Divine Husbandman's Classic off Materia Medica (1625); and Bensky et al, Chinese Herbal Medicine, Materia Medica, Eastland Press, Seattle, Wash. (1993)). Formulations have been carefully crafted so as to accentuate and enhance functionality, to suppress and counteract toxicity, and to avoid antagonism and incompatibility. Since there are so many possible combinations from such a large collection of herbs, a hierarchical scheme has been developed. That is, the principal ingredient is a substance that provides the main therapeutic thrust, the second principal ingredient enhances or assists the therapeutic actions of the first, and the rest of the ingredients serve one or more of the following functions: treatment of accompanying symptoms, moderation of the harshness or toxicity of the primary substances, guidance of the medicine to the proper organs or exertion of a harmonizing effect.
III. Herbs with Antimicrobial Activities Described in the Chinese Pharmacopoeia
Many of the 5767 Chinese medicines (herbs, animals and minerals) listed in the Encyclopedia of Traditional Chinese Medicinal Substances (1977) have been used to combat microbial infections.
The herb Zi-Cao (literally translated as "Purple Herb"), is the dry root of Lithospermrum erythrorhizon Sieb, et Zucc. or Arnebia euchroma (Royle) Johnst. These plants belong to the family Boraginaceae. This herb is officially listed in the Chinese Pharmacopoeia, and has frequently been used as an anti-inflammatory and anti-pyretic agent in the treatment of measles, eczema and thermal burns (Tang et al, Chinese Drugs of Plant origin, pages 613-619, Springer-Verlag (1992)). The aqueous or organic extract of these roots has been used in combination with other herbs in the form of ointments for topical use, or in the form of aqueous infusions as an antipyretic to "cool the blood and as an antidote to body toxins induced by heat excess".
The roots of plants from the family of Boraginaceae contain naphthoquinone pigments as the main chemical constituents (Tang et al, supra). Shikonin and its derivatives are the main naphthoquinone pigments. Shikonin is a naphthoquinone with an unsaturated side chain and an asymmetric centrum bearing a hydroxy group. A series of carboxylic acid esters of shikonin have been identified (Morimoto et al, Tetrahedron Lett., 52:4737-4739 (1965); Morimoto et al, Tetrahedron Lett., 31:3677-3680 (1966); and Kyogoku et al, Shoyakugagu Zasshi, 27:24-30 (1973)). The chemical structures of shikonin and its identified derivatives are shown in FIG. 1.
The total organic solvent extract from root of a plant belonging to the family Boraginaceae, and some of the individual components of the extract, i.e., shikonin and deoxyshikonin, have been identified as having antimicrobial activity (Tanaka et al, Yakugaku Zasshi, 92:525-530 (1972); Kyogoku et al, Shoyakugaku Zasshi, 27:31-36 (1973); and Honda et al, J. Natural Products, 51:152-154 (1988)); and anti-inflammatory activity (Tanaka et al, J. Natural Products, 9:466-469 (1986)). However, there is no teaching or suggestion in the art that the extracts, or components thereof, are effective against antibiotic-resistant bacteria, much less gram-positive antibiotic-resistant bacteria.